J. Anat. (1990), 168, 137-141
137
With 5 figures Printed in Great Britain
Prolactin receptors in the primate choroid plexus RAYMOND J. WALSH, LUZ P. MANGURIAN AND BARRY I. POSNER*
Department of Anatomy, George Washington University Medical Center, Washington D.C. 20037, USA and *Department of Medicine, Royal Victoria Hospital, Montreal, Quebec, Canada
(Accepted 27 June 1989) INTRODUCTION
While the interactions of pituitary-derived prolactin with the central nervous system have been extensively described in lower vertebrates, prolactin interactions with the primate central nervous system have received less attention. Although it is tempting to extrapolate the results from lower mammals to higher vertebrate forms, potential variations up the evolutionary scale cannot be dismissed. The acquisition of monkey choroid plexus provided the opportunity to examine the primate choroid plexus for the presence of specific prolactin binding sites, sites that have been shown to exist in the choroid plexus of lower vertebrate forms. MATERIALS AND METHODS
Choroid plexus was obtained from three sexually mature, male Cynomolgus monkeys immediately after killing with an intravascular injection of T61 Euthanasia Solution (Hoechst Roussell Agri-Vet, Somerville, NJ). The choroid plexus was removed from the lateral and fourth ventricles, placed in 0-25 M sucrose and frozen at -20 °C until use. The detection of specific prolactin binding sites in the choroid plexus was performed with an in vitro autoradiographic technique. All hormones were obtained from the National Hormone & Pituitary Program of the National Institute of Diabetes, Digestive and Kidney Disease, the Center for Population Research of the National Institute of Child Health and the Agricultural Research Service of the U.S. Department of Agriculture. Ovine prolactin was iodinated with the chloramine-T method and purified either by column chromatography or dialysis. The specific activity of the radiolabelled prolactin was 160 ,cCi/mg (n = 2). The choroid plexus was thawed on crushed ice and centrifuged at 300 rpm for 20 seconds. The choroid plexus pellets were embedded in 0. C. T. tissue embedding compound at -20 'C. Sixteen micrometer thick cryostat sections were placed on cleaned glass slides. Tissue sections were preincubated at 4 'C in 25 mm Tris-HCl (pH 7-4), 10 mm MgCl2, 0-1 % BSA for four minutes. Sections were incubated overnight (18 hours) at 4 'C in the same medium containing 17 2 ng of I'l25-prolactin alone (total binding) or with a 500-fold excess of either unlabelled ovine prolactin, bovine growth hormone, or ovine luteinizing hormone (non-specific binding). An additional series of sections was incubated with 17-2 ng of [125I]prolactin containing various concentrations of excess unlabelled prolactin, i.e. 85 ng, 430 ng, 2150 ng, 4300 ng.
138
R. J. WALSH, L. P. MANGURIAN AND B. I. POSNER
Following incubation, the sections were rinsed in two changes of buffer and two changes of distilled water, 4 minutes each. The sections were air dried, apposed to Kodak XAR-5 film, and exposed at room temperature for four days. Films were developed in Kodak D-19 for 5 minutes and fixed with Kodak fixative. The autoradiographic reactions were quantified with an RAS-GN1000 Research Analysis System (Amersham Corp.). Three sections were quantified per animal for both the specificity and dose-response studies. Data were subjected to the Student's t test to determine statistical significance.
RESULTS
Cryostat sections stained with 0 5 % aqueous thionin revealed the characteristic villi of the choroid plexus, which are covered by a single layer of epithelial cells (Fig. 1). Sections incubated with [125I]prolactin alone (total binding) produced an intense autoradiographic reaction on the XAR-5 film (Fig. 2). The autoradiographic reaction was visually reduced in sections co-incubated with excess unlabelled prolactin (nonspecific binding; Fig. 3). Quantitation of the autoradiographs revealed a 64% displacement (n =3) of radiolabelled prolactin by the excess unlabelled prolactin (P
137
With 5 figures Printed in Great Britain
Prolactin receptors in the primate choroid plexus RAYMOND J. WALSH, LUZ P. MANGURIAN AND BARRY I. POSNER*
Department of Anatomy, George Washington University Medical Center, Washington D.C. 20037, USA and *Department of Medicine, Royal Victoria Hospital, Montreal, Quebec, Canada
(Accepted 27 June 1989) INTRODUCTION
While the interactions of pituitary-derived prolactin with the central nervous system have been extensively described in lower vertebrates, prolactin interactions with the primate central nervous system have received less attention. Although it is tempting to extrapolate the results from lower mammals to higher vertebrate forms, potential variations up the evolutionary scale cannot be dismissed. The acquisition of monkey choroid plexus provided the opportunity to examine the primate choroid plexus for the presence of specific prolactin binding sites, sites that have been shown to exist in the choroid plexus of lower vertebrate forms. MATERIALS AND METHODS
Choroid plexus was obtained from three sexually mature, male Cynomolgus monkeys immediately after killing with an intravascular injection of T61 Euthanasia Solution (Hoechst Roussell Agri-Vet, Somerville, NJ). The choroid plexus was removed from the lateral and fourth ventricles, placed in 0-25 M sucrose and frozen at -20 °C until use. The detection of specific prolactin binding sites in the choroid plexus was performed with an in vitro autoradiographic technique. All hormones were obtained from the National Hormone & Pituitary Program of the National Institute of Diabetes, Digestive and Kidney Disease, the Center for Population Research of the National Institute of Child Health and the Agricultural Research Service of the U.S. Department of Agriculture. Ovine prolactin was iodinated with the chloramine-T method and purified either by column chromatography or dialysis. The specific activity of the radiolabelled prolactin was 160 ,cCi/mg (n = 2). The choroid plexus was thawed on crushed ice and centrifuged at 300 rpm for 20 seconds. The choroid plexus pellets were embedded in 0. C. T. tissue embedding compound at -20 'C. Sixteen micrometer thick cryostat sections were placed on cleaned glass slides. Tissue sections were preincubated at 4 'C in 25 mm Tris-HCl (pH 7-4), 10 mm MgCl2, 0-1 % BSA for four minutes. Sections were incubated overnight (18 hours) at 4 'C in the same medium containing 17 2 ng of I'l25-prolactin alone (total binding) or with a 500-fold excess of either unlabelled ovine prolactin, bovine growth hormone, or ovine luteinizing hormone (non-specific binding). An additional series of sections was incubated with 17-2 ng of [125I]prolactin containing various concentrations of excess unlabelled prolactin, i.e. 85 ng, 430 ng, 2150 ng, 4300 ng.
138
R. J. WALSH, L. P. MANGURIAN AND B. I. POSNER
Following incubation, the sections were rinsed in two changes of buffer and two changes of distilled water, 4 minutes each. The sections were air dried, apposed to Kodak XAR-5 film, and exposed at room temperature for four days. Films were developed in Kodak D-19 for 5 minutes and fixed with Kodak fixative. The autoradiographic reactions were quantified with an RAS-GN1000 Research Analysis System (Amersham Corp.). Three sections were quantified per animal for both the specificity and dose-response studies. Data were subjected to the Student's t test to determine statistical significance.
RESULTS
Cryostat sections stained with 0 5 % aqueous thionin revealed the characteristic villi of the choroid plexus, which are covered by a single layer of epithelial cells (Fig. 1). Sections incubated with [125I]prolactin alone (total binding) produced an intense autoradiographic reaction on the XAR-5 film (Fig. 2). The autoradiographic reaction was visually reduced in sections co-incubated with excess unlabelled prolactin (nonspecific binding; Fig. 3). Quantitation of the autoradiographs revealed a 64% displacement (n =3) of radiolabelled prolactin by the excess unlabelled prolactin (P
